Interpretive Summary: Huanglongbing (HLB – formerly known as “citrus greening”) is one of the most destructive diseases of citrus in Florida and other citrus-producing countries worldwide. In Florida the disease is associated with the bacterium Candidatus Liberibacter asiaticus (Las), which enters the plants via a sap-feeding insect, the Asian citrus psyllid. Unlike other bacteria, Las cannot be cultured in the laboratory at present, and researchers have to rely on the insect to infect citrus plants for their experiments, which is logistically demanding. An easier method to infect citrus with Las is by grafting with tissue from infected plants. The purpose of this study was to find out if these two methods of artificial infection of citrus with Las result in similar frequencies of infection and a similar pattern of HLB disease development. We also compared the two methods with the natural progression of the disease in the field. Six greenhouse experiments were conducted with HLB-susceptible and HLB-tolerant plants and frequencies of transmission of Las varied between experiments and depended on the plant type. In susceptible sweet orange plants Las was transmitted to up to 90 percent of plants after six to 12 months using the grafting method. In tolerant US-802 plants Las was transmitted with a frequency of 31 to 75 percent. In contrast, Las was transmitted to no more than 38 percent of plants using the psyllid-inoculation method, although the speed of Las transmission was faster in tolerant US-802 plants. HLB disease symptoms were indistinguishable in graft- and psyllid-inoculated citrus plants. Compared with controlled infection in the greenhouse experiments, natural infection of field-grown sweet orange trees occurred at a much slower pace requiring more than one year for 50 percent of trees to become infected with Las and a minimum of three years for 100% of trees to become infected.

Technical Abstract:
Candidatus Liberibacter asiaticus (Las) is a phloem-limited bacterium associated with Huanglongbing (HLB), one of the most destructive diseases of citrus in Florida and other citrus-producing countries. Natural transmission of Las occurs by the psyllid vector Diaphorina citri, but transmission can also occur through grafting with diseased budwood. Due to the difficulty of maintaining Las in culture, screening of citrus germplasm for HLB resistance often relies on graft-inoculation as the mode of pathogen transmission. This study evaluates transmission efficiencies and HLB progression in graft-inoculated and psyllid-inoculated citrus under greenhouse- and natural conditions in the field. Frequencies of transmission in graft-inoculated greenhouse-grown plants varied between experiments and were as high as 90% in susceptible sweet orange plants six to 12 months after inoculation. Transmission frequency in a more tolerant Citrus x Poncirus genotype (US-802) was 31 to 75 percent. In contrast, transmission of Las after controlled psyllid inoculation did not exceed 38% in any of four experiments in this study. Whereas transmission of Las was faster through psyllids in US-802 plants, the speed of transmission was similar after graft- and psyllid inoculation in sweet orange plants. HLB symptom expression was indistinguishable in graft- and psyllid-inoculated plants, but was not always associated with the number of bacteria in affected leaves. The highest number of Las genomes per gram leaf tissue measured in sweet orange plants was 1-4 x 107 in graft-inoculated plants and 1-2 x 107 in psyllid-inoculated plants. Highest numbers measured in tolerant US-802 plants were 1-3 x 106 and 2-6 x 106, respectively. Compared with artificial inoculation in a greenhouse setting, natural inoculation of field-grown sweet orange trees occurred at a much slower pace, requiring more than one year for infection incidence to reach 50% and a minimum of three years to reach 100 percent.